Francisella tularensis is a facultative intracellular gram-negative bacterium responsible for human tularemia. Interest in the pathogen has increased recently with the realization that it could be used as an agent of bioterrorism due to its high infectivity, potentially high mortality rates and the ability to deliver the organism to large numbers of persons by aerosol. Natural infections with F. tularensis proceed from an initial localized site via blood dissemination to the spleen, liver, lungs, CMS and kidneys. Immunity sufficient to resolve primary infections and mediate memory responses to secondary exposure is dependent on T cells, the cytokine interferon-gamma (IFN-g) and the activation of macrophages. However, relatively little is known about protective immune responses to primary infection that occur within the first week of exposure. Thus, it is not known which cells of the innate immune system respond to the bacterium, how they recognize microbial components or how they might initiate clearance. The long-term objective of this research is to characterize the cellular basis of IFN-g production during the initial phase of F. tularensis infection and define the mechanisms of immune recognition of the microbe in naive mice. Our central hypothesis is that IFN-g production by NK and NKT cells, which recognize the bacterium through their Toll-like receptors (TLR), is a key process in the early protective immunity. Our specific aims are to: (i) identify the cellular subsets that are activated for IFN-g production during F. tularensis infection; (ii) define the role of TLR and accessory signals in the induction of these responses; and (iii) determine the role of TLR-mediated NK and NKT cell activation in early clearance of F. tularensis. Besides providing important insight into the nature of innate immunity to F. tularensis, this R21 application will assess the participation of NK/NKT cells and TLR-mediated signaling in these protective responses and generate important preliminary data to characterize the role of these cells in immune protection during the first few days of infection.